Homology of the primary structures of cytoplasmic and mitochondrial aspartate aminotransferases from pig heart

Doonan, Shawn; Hughes, Graham J.

doi:10.1016/0014-5793(74)80623-x

Cited by 23 publications

(6 citation statements)

References 12 publications

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“…The sheep liver isoenzymes have similar coenzymebinding peptides (Campos-Cavieres & Milstein, 1975), so it seems likely that they have similar sequences in all those portions of their chains that constitute the active site. This contention holds for pig heart aspartate aminotransferase isoenzymes (Doonan et al, 1974). Despite this similarity some difference must exist between the micro-environment of the active site of cytoplasmic and mitochondrial aspartate aminotransferase from sheep liver to account for the differences in their kinetic properties.…”

Section: Discussionmentioning

confidence: 99%

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver

Orlacchio¹,

Campos-Cavieres²,

Pashev³

et al. 1979

Biochemical Journal

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A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.

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Section: Discussionmentioning

confidence: 99%

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver

Orlacchio¹,

Campos-Cavieres²,

Pashev³

et al. 1979

Biochemical Journal

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“…This enzyme is of particular interest since it exists in two molecular forms in the cells of most higher organisms (Boyd, 1961;Wada & Morino, 1964), one associated with the soluble fraction and the other with the mitochondria; Baumber & Doonan (1976) have shown that the localization of the two forms is unique in that no mitochondrial isoenzyme is detectable in the cytoplasmic fraction from rat liver and vice versa. The two isoenzymes from any particular organism differ widely in chemical, physical, catalytic and immunochemical proper- ties (Wada & Morino, 1964), but it has been shown (Doonan et al, 1974) that the structures of the isoenzymes from pig heart muscle are homologous. Little is known about the biosynthesis of the mitochondrial isoenzyme, but studies with somatic cell hybrids (van Heyningen et al, 1974) have shown that it is coded by the nuclear genome and is therefore probably synthesized on cytoplasmic ribosomes.…”

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confidence: 99%

Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro

Marra¹,

Doonan²,

Saccone³

et al. 1977

Biochemical Journal

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1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.

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“…Where two or more distinct gene loci, each coding for a structurally distinct polypeptide chain, are concerned in determining the structures of a set of isozymes with the same or very similar catalytic properties, it is usually thought that such multiple loci have arisen by gene duplication and have subsequently diverged through independent mutational events. That this is true for at least one mitochondrial-soluble enzyme pair, the glutamate-oxaloacetate transaminases, has been confirmed by primary structure analysis (see Doonan et al 1975) for the complete structure of the soluble enzyme and Doonan et al (1974) for a partial structure of the mitochondrial form). The observation that aconitase, together with many other mitochondrial-soluble pairs of enzymes, has a relatively more basic mitochondrial form and a relatively more acidic soluble form implies a statistically highly significant tendency for amino acid substitution in the ' mitochondrial ' evolutionary lines of descent to confer increasing basicity on the enzyme protein and for amino acid substitutions in the 'soluble ' lines to confer increasing acidity.…”

Section: Isoelectric Points and Electrophoretic Mobilitiesmentioning

confidence: 90%

The distribution and properties of aconitase isozymes in man

1977

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SUMMARY Electrophoretic analysis of sub cellular fractions prepared from rat and mouse liver indicate that the more acidic group of aconitase isozymes represents the soluble enzyme whilst the more basic group represents the mitochondrial form. The corresponding groups of isozymes in man can be similarly identified by analogy with the rodent patterns. Qualitatively similar isozyme patterns are found in a variety of human tissues during both adult and foetal life, but the tissues vary one from another both in the total levels of aconitase activity they contain and in the relative activities of the soluble and mitochondrial enzyme. During foetal development each isozyme undergoes an increase in activity in several tissues, although the relative activities in some tissues change during gestation. Investigation of the substrate specificities of the isozymes using activity stains designed to follow the enzymic conversion of citrate and cia‐aconitate to isocitrate, and of isocitrate and cis‐aconitate to citrate, reveal no evidence for the existence of separate α‐ and β‐aconitases. Isoelectric focusing under standard conditions yields an isoelectric point value of 6.9 5 0.2 for the human mitochondrial aconitase and a value of 6.1 ± 0.1 for the soluble aconitase. Gel filtration either in the presence or absence of substrate yields a molecular weight value of 65000 ± 4000 for the human mitochondrial aconitase and a value of 83000 ± 6000 for the soluble aconitase. A difference in molecular weights of this magnitude is unusual amongst systems of isozymes and is of interest with regard to the evolutionary history of aconitase. Investigation of the relative heat stabilities of the isozymes both by electrophoretic analysis of heated aqueous tissue extracts and by heating the isozymes in starch gels after electrophoretic separation at pH 7.4 indicates that the soluble aconitase is more stable than the mitochondrial. The isozymes of mitochondrial aconitase are found to give rise to secondary, more anodal components upon storage of aqueous tissue extracts at 4–6 °C. The electrophoretic pattern given by the soluble aconitase, however, remains unchanged under similar conditions.

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Homology of the primary structures of cytoplasmic and mitochondrial aspartate aminotransferases from pig heart

Cited by 23 publications

References 12 publications

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver

Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro

The distribution and properties of aconitase isozymes in man

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